Carbapenems are a class of β-lactam antibiotics with a broad spectrum of antibacterial activity. Carbapenem antibiotics were originally developed from thienamycin, a naturally derived product of Streptomyces cattleya. They have a structure that is highly resistant to most β-lactamases and consequently, are often the antibiotic of last resort for treatment of highly resistant infections of bacteria such as Escherichia coli and Klebsiella pneumunia. Carbapenem antibiotics include, but are not limited to, imipenem, ertapenem, meropenem, doripenem, panipenem, biapenem, razupenem, and tabipenem.
Imipenem, (compound (1) shown below), is a carbapenem that exhibits a broad range of antibiotic activity against gram-positive and gram-negative aerobic and anaerobic bacteria species.

The methods of use and manufacture of imipenem were first disclosed in U.S. Pat. No. 4,194,047. Various alternative synthetic methods for the preparation of imipenem are described in e.g., U.S. Pat. Nos. 4,374,772, 4,894,450, and 7,462,712. Several synthesis routes for the preparation of imipenem include the formation of a p-nitrobenzyl-ester of imipenem (compound (2) shown below).

This pNB-protected imipenem of compound (2) is deprotected to provide the imipenem product of compound (1) by using palladium or platinum hydrogenation catalysts to remove the remove p-nitrobenzyl group (see e.g., U.S. Pat. Nos. 4,292,436, and 5,245,069, and U.S. Pat. Publ. 2002/0095034). The use of these catalysts for pNB deprotection, however, is expensive and results in relatively low yields of the deprotected imipenem (e.g., <60%).
The use of an esterase enzyme to remove pNB protecting groups in the synthesis of the cephalosporin-derived and 1-carba-cephalasporin antibiotics was disclosed in U.S. Pat. No. 5,468,632. A specific wild-type pNB esterase from Bacillus subtilis for removing such pNB protecting groups was isolated, cloned, and sequenced in U.S. Pat. No. 5,468,632. This same wild-type pNB esterase also has been engineered for increased thermostability and activity in the removal of the pNB group from a pNB-protected precursor of the antibiotic Loracarbef (see e.g., Moore et al., “Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents,” Nature Biotechnology 14: 458-467 (1996); Moore et al. “Strategies for the in vitro Evolution of Protein Function: Enzyme Evolution by Random Recombination of Improved Sequences.” J. Mol. Biol. 272:336-347 (1997); Giver et al., “Directed evolution of a thermostable esterase.” Proc. Natl. Acad. Sci. USA 95: 12809-12813 (October 1998). There remains, however, a need for an engineered pNB esterase that provides selectivity and high yields in the deprotection of pNB-protected carbapenem intermediates, such as the pNB-protected imipenem of compound (2), under commercially viable and industrially useful process conditions.